Nucleoside phosphorylases, which can be found in bacteria and other organisms, catalyze the reversible phosphorolysis of nucleosides and the transferase reaction involving purine and pyrimidine bases. Purine and pyrimidine nucleoside phosphorylases have been isolated from a number of bacterial cells. Utagawa et al., FEBS Lett. 109: 261-63 (1980); Utagawa, et al., Agri. Biol. Chem. 49: 3239-46 (1985); Engelbrecht, et al., J. Biol. Chem. 244: 6228-32 (1969); Jensen, Eur. J. Biochem. 61: 377-86 (1976); Jensen, et al., Eur. J. Biochem. 51: 253-65 (1975). For example, uridine phosphorylase, thymidine phosphorylase and purine nucleoside phosphorylase have been purified from Escherichia coli. Krenitsky et al., Biochemistry 20: 3615-21 (1981); Schwartz, et al., Eur. J. Biochem. 21: 191-98(1971). Additionally, a thermostable purine nucleoside phosphorylase enzyme has been isolated from Bacillus stearothermophilus. Saunders, et al., J. Biol. Chem. 244. 3691-97 (1969); Hori et al., Agric. Biol. Chem. 53: 2205-2210 (1989).
These nucleoside phosphorylases possess fairly broad substrate specificity and have been utilized in the synthesis of natural and modified ribonucleoside analogs. Shirae et al., Agric. Biol. Chem. 52: 1499-1504 (1988); Hennen et al., J. Org. Chem. 54:4692-95 (1989); Hori et al., Agric. Biol. Chem. 55: 1071-1074 (1991); Hori et al., J. Biotech. 17: 121-131 (1991); Hori et al., Biosci. Biotech. Biochem. 56: 580-582 (1992). There are fewer examples of the use of whole cells in the synthesis of nucleosides, particularly 2'-deoxynucleosides. Morisawa et al., Tetrahedron Lett. 21: 479-482 (1980); Holy et al., Nucleic Acid Research Symposia 18:69-72 (1987). Morisawa et al. used a combination of Enterobacter aerogene cells and chemical steps to synthesize arabinofuranosyl purine nucleosides. Holy et al. used E. coli cells and 2'-deoxyuridine as substrate to synthesize 2'-deoxy purine and pyrimidine nucleosides, but E. coli cannot be used above physiological temperatures.
Several nucleosides have important diagnostic and therapeutic uses. For example, the natural 2'-deoxynucleoside, thymidine, is a precursor for the synthesis of a number of important anti-HIV active nucleosides such as 3'-deoxy-3'azidothymidine (AZT) and dideoxydidehydrothymidine (d4T) and various potential antiviral compounds. Thymidine and other deoxynucleosides (both natural and non-natural) are precursors of certain oligomers referred to as "anti-sense oligonucleotides," many of which are being investigated as drugs against viruses, tumors and bacterial infections.
Yet thymidine and other deoxynucleosides are relatively expensive and difficult to obtain by known approaches. For example, prior to the present invention, thymidine was produced by the hydrolysis of DNA derived from natural sources, like milt, but there are limitations using this approach. Dekker et al., J. Chem. Soc. 947-55 (1953). Chemical methods also are available for the synthesis of thymidine, but these involve complex, multi-step methodologies.
For example, an early chemical synthesis of thymidine involves a multi-step procedure which gave very low overall yields of thymidine (&lt;5%). Shaw et al., Proc. Chem. Soc. 81-82 (1958); Shaw et al., J. Chem. Soc. 50-55 (1959). A more direct chemical method involves coupling of suitably protected thymine with an .alpha.-chloro-2-deoxy sugar. Hubbard et al., Nucleic Acids Res. 12: 6827-36 (1984). While the coupling step proceeds in good yields, the desired .beta.-thymidine derivative is produced together with its .alpha.-isomer and separation of the desired .beta.-isomer requires extensive chromatography. In addition, suitably protected bases have to be prepared for the coupling. Also, the .alpha.-chloro sugar for the coupling reaction is unstable and has to be carefully prepared and handled. Thus, the overall yield of thymidine by this method is low. A multi-step synthesis of thymidine from D-xylose also has been reported. Rao et al., Proc. Ind. Acad. Sci. (Chem. Sci.) 106:1415-21 (1994). However, the overall yield in this case is only 24% and the synthesis involves the use of some difficult to handle reagents (e.g. SnCl.sub.4). Morisawa et al., Tetrahedron Lett. 21: 479-482 (1980) used a combination of Enterobacter aerogene cells and chemical steps to synthesize a few arabinofuranosyl purine nucleosides. However, the yield in the enzymatic step alone was merely 34% and only arabinofuranosyl purine nucleosides were synthesized. Holi et al., Nucleic Acid Research Symposia 18: 69-72 (1987) used E. coli cells and only 2'-deoxyuridine as substrate to synthesize only 2'-deoxy purine and pyrimidine nucleosides. Holi et al. reported that the % conversion to thymidine was 67% in their synthesis. The disadvantage of the approach of Holi et al. include the use of 2'-deoxyuridine, which is expensive relative to other substrates. Additionally, their reliance upon E. coli limits the reaction temperature, which leads to slower reaction kinetics and decreased solubilities of reactants and products.
Accordingly, there is a need for improved methods for producing thymidine and other deoxynucleosides that have broad range applications, including the production of other natural and non-natural nucleosides. Due to the societal importance of these nucleosides, there is needed methods capable of being undertaken on an industrial scale. These needs have been unresolved until the advent of the present invention.